PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is uniquely difficult to treat due to late diagnosis and limited medical management options for a majority of patients. This is despite a wealth of watershed studies on disease driver mechanisms over the past decade, highlighting a need for alternative approaches to studying this disease. It is now clear that PDACs present along a bimodal continuum of transcriptomic subtypes that exhibit distinct prognoses, and additional work on advanced PDAC models has uncovered that these tumors, though highly chemoresistant, are sensitive to external amino acid supply through metabolic dysregulation. Transcripts that define known PDAC molecular subtypes present unique codon biases, suggesting that PDACs are subject to biologically deterministic codon-level selective pressures. Work from our lab and others has shown that the abundance of properly aminoacylated transfer RNAs (tRNAs), highly structured and chemically modified non- coding RNAs, is highly deterministic of mRNA half-life. This mechanism acts through codon-anticodon recognition and ribosome elongation rate, and alterations of functional tRNA abundance can dictate cellular functions via concomitant regulation of mRNA stability. Furthermore, a wealth of literature evidence in diverse cell types across species demonstrates that tRNA regulation can be disease-specific. Thus, tRNAs may likely serve a key regulatory role in PDAC subtype expression and amino acid sensitivity, given their function in bridging codon-amino acid pairings during protein production. As proof of concept, I have strong preliminary evidence that tRNA expression can be highly predictive of disease stage in a limited cohort of primary colorectal tumor samples. Furthermore, a pilot study of PDACs revealed widespread tRNA dysregulation, with increased use of cysteine-decoding transcripts, matching existing literature that PDACs are specifically sensitive to deprivation of this amino acid. In this proposal, I seek to interrogate linked roles of tRNAs as regulators of mRNA and nutrient availability phenotypes in PDAC. My central hypothesis is that PDACs specifically regulate tRNA expression to confer cell survival and proliferation advantages and that tRNA profiling can reveal novel biomarkers for use in clinical decision-making. I will address this hypothesis through the following aims: Aim 1: Characterize tRNA expression and modifications in primary patient PDACs; Aim 2: Investigate PDAC tRNA regulation as a driver of tumor cell survival. These aims will be achieved through a combination of biochemical and high-throughput sequencing approaches using archived patient samples and established in vitro cell lines. Beyond biological interrogation, this proposal involves novel technical development in the experimental and analytical application of tRNA sequencing for large patient sample cohorts. This project would be the first to analyze tRNA gene-specific regulation in cancer, and wil...